Peptide repeat immunogens

ABSTRACT

The invention relates to a polypeptide including (1) a receptor binding domain of a  Pseudomonas  exotoxin A, and (2) at least two copies of a peptide sequence.

BACKGROUND OF THE INVENTION

The successful development of a protein-based vaccine often requires adelicate balance between enhancing immunogenicity of a particularantigen and the potential toxicity elicited by such enhancing. Forexample, an effective adjuvant used in animal studies (e.g., completeFreund's) may be too toxic to be used in vaccines prepared for humans.

SUMMARY OF THE INVENTION

The invention is based on the discovery of a new means of generating animmune response to a peptide antigen by concatemerizing the peptide andfusing the concatemer to a receptor binding domain of a Pseudomonasexotoxin. Such a fusion protein elicits antigen-specific antibodies in avariety of mammals, with little or no toxicity observed.

Accordingly, the invention features a polypeptide including (1) at leasttwo copies (e.g., at least 12 or 16 copies) of a peptide sequence, and(2) a receptor binding domain of a Pseudomonas exotoxin A.

The peptide sequence must be at least two amino acids in length (e.g.,at least 3, 5, 7, 9, or 10 amino acids in length). In a preferredembodiment, the peptide sequence can be less than 1000 amino acids inlength (e.g., less than 500, 100, 50, or 20 amino acids in length). Thepeptide sequence can include any antigen in which an immune responseagainst it is beneficial, such as gonadotropin releasing hormone or GnRH(e.g., EHWSYGLRPG [SEQ ID NO:1]) or a fragment of a vaccinia virus coatprotein (e.g., LIGICVAVTVAI [SEQ ID NO:2]).

A receptor binding domain is an amino acid sequence within a polypeptidewhich specifically binds to a LDL/alpha₂-microglobulin cell receptor. Anexample of a receptor binding domain is amino acids 1-252 (domain Ia) ofthe P. aeruginosa exotoxin A (PE):

-   -   mhliphwiplvaslgllaggssasaaeeafdlwnecakacvldlk        dgvrssrmsvdpaiadtngqgvlhysmvleggndalklaidnals        itsdgltirleggvepnkpvrysytrqargswslnwlvpighekp        snikvfihelnagnqlshmspiytiemgdellaklardatffvra        hesnemqptlaishagvsvvmaqtqprrekrwsewasgkylclld        pldgvynylaqqrcnlddtwegkiyrv (SEQ ID NO:3).        Variants of the sequence immediately above, including        substitutions, deletions, or additions are permissible, provided        that the receptor binding domain specifically binds to a        LDL/alpha₂-microglobulin cell receptor.

The position of the various elements within the polypeptide can bevaried, as long the polypeptide is still able to elicit an immuneresponse in a mammal. For example, all copies of the peptide sequencecan be in a consecutive series, meaning that all copies occur as asingle block of peptide repeats without intervening amino acids inbetween any two copies. In addition, the receptor binding domain canoccur anywhere in the polypeptide (e.g., at the N-terminus, at theC-terminus, internal, or between two copies of the peptide sequence), aslong as the intended function of the polypeptide as an antigen is notdisrupted.

The invention also includes nucleic acids (e.g., expression vectors,including viral vectors) that encode a polypeptide of the invention.Such nucleic acids can be used in a naked DNA-based vaccines or toproduce the polypeptides of the invention in large quantities.

In addition, the invention features a method of producing a polypeptideby (1) providing a nucleic acid of the invention, (2) introducing thenucleic acid into a cell (e.g., a bacteria or eukaryotic cell, includingcell lines and primary cells), and (3) expressing the polypeptide in thecell.

The polypeptides and nucleic acids of the invention can be used toprovide new antigens and vaccines for eliciting an immune responseagainst specific peptide sequences. These antigens can be formulated assafe and effective vaccines in mammals and humans, or at the very least,tested for efficacy in human and/or animal models, including primateanimal models.

Other features or advantages of the present invention will be apparentfrom the following drawings and detailed description, and also from theclaims.

BRIEF DESCRIPTION OF THE DRAWING

FIG. 1 is a representation of a GnRH peptide sequence (SEQ ID NO:1)useful in the polypeptides of the invention and primer sequences (SEQ IDNO:4 for Oligo A; SEQ ID NO:5 for Oligo B) which encode it or portionsthereof (DNA sequence A1 and A2 is designated SEQ ID NO:4; DNA sequenceB1 and B2 is designated SEQ ID NO:12).

FIG. 2 is a representation of how successive extension, denaturation,and annealing (i.e., PCR) of the primers in FIG. 1 produce a nucleicacid encoding concatamers of the peptide sequence.

FIG. 3 is a representation of a final PCR amplification of theconcatemeric nucleic acid shown in FIG. 2 to introduce at the ends ofthe concatemer a stop codon and suitable restriction sites for cloning.Adaptor primer A is designated SEQ ID NO:6. Adaptor primer B isdesignated SEQ ID NO:7. The 5′ overhang sequence containing the EcORIsite is designated SEQ ID NO:8 (complimentary strand is designated SEQID NO:11). The 5′ overhang sequence containing the SacII site isdesignated SEQ ID NO:9 (complimentary strand is designated SEQ IDNO:10).

DETAILED DESCRIPTION

The invention relates to new polypeptides having multiple copies of apeptide antigen fused to the receptor binding domain of a Pseudomonasexotoxin. Multiple copies of a peptide antigen is believed to increaseimmunologic presentation of epitopes present in each monomeric peptidesequence, though the mechanism is unclear. In addition, the exotoxinfragment is believed to increase binding between the polypeptide andantigen presenting cells, thereby facilitating uptake and presentationof peptide sequences.

I. Generation of Antibodies

The polypeptides of the invention can be used to generate antibodiesthat specifically bind a monomeric peptide sequence. Such antibodies canbe used in diagnostic and/or therapeutic procedures that require theenhancement, inhibition, or detection of any molecule which contains theepitope presented by the peptide sequence.

In particular, various host animals can be immunized by injection of acomposition containing a polypeptide of the invention. Host animals caninclude rabbits, mice, guinea pigs, and rats. Various adjuvants can beused to increase the immunological response, depending on the hostspecies, including but not limited to Freund's (complete andincomplete), mineral gels such as aluminum hydroxide, surface activesubstances such as lysolecithin, pluronic polyols, polyanions, peptides,oil emulsions, keyhole limpet hemocyanin, dinitrophenol, and potentiallyuseful human adjuvants such as BCG (bacille Calmette-Guerin) andCoxynebacterium parvum.

Antibodies include polyclonal antibodies, monoclonal antibodies,humanized or chimeric antibodies, single chain antibodies, Fabfragments, F(ab′), fragments, and molecules produced using a Fabexpression library.

Monoclonal antibodies, which are homogeneous populations of antibodiesto a particular antigen, can be prepared using the polypeptidesdescribed above and standard hybridoma technology (see, e.g., Kohler etal., Nature 256:495, 1975; Kohler et al., Eur. J. Immunol. 6:511, 1976;Kohler et al., Eur. J. Immunol. 6:292, 1976; Hammerling et al., In:Monoclonal Antibodies and T Cell Hybridomas, Elsevier, N.Y., 1981; U.S.Pat. No. 4,376,110; Kosbor et al., Immunology Today 4:72, 1983; Cole etal., Proc. Natl. Acad. Sci. USA 80:2026, 1983; and Cole et al.,Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp. 77-96,1983). Such antibodies can be of any immunoglobulin class including IgG,IgM, IgE, IgA, IgD and any subclass thereof. The hybridoma producing theMab of this invention may be cultivated in vitro or in vivo. The abilityto produce high titers of mAbs in vivo makes this an excellent method ofproduction.

The antibodies can be used, for example, to detect the presence of anantigen in a biological sample as part of a diagnostic assay, and alsoto evaluate the effectiveness of medical treatments by other therapeuticapproaches.

In addition, techniques developed for the production of “chimericantibodies” (Morrison et al., Proc. Natl. Acad. Sci. USA 81:6851, 1984;Neuberger et al., Nature 312:604, 1984; Takeda et al., Nature 314:452,1984) by splicing the genes from a mouse antibody molecule ofappropriate antigen specificity together with genes from a humanantibody molecule of appropriate biological activity can be used. Achimeric antibody is a molecule in which different portions are derivedfrom different animal species, such as those having a variable regionderived from a murine Mab and a human immunoglobulin constant region.

Alternatively, techniques described for the production of single chainantibodies (U.S. Pat. Nos. 4,946,778, 4,946,778, and 4,704,692) can beadapted to produce single chain antibodies against a particular peptideantigen. Single chain antibodies are formed by linking the heavy andlight chain fragments of the Fv region via an amino acid bridge,resulting in a single chain polypeptide.

Antibody fragments that recognize and bind to specific epitopes can begenerated by known techniques. For example, such fragments include, butare not limited to, F(ab′)₂ fragments that can be produced by pepsindigestion of the antibody molecule, and Fab fragments that can begenerated by reducing the disulfide bridges of F(ab′)₂ fragments.

II. Production and Use of Vaccine Compositions

The invention includes vaccine compositions (e.g., parenteral injectablevaccines) containing at least one polypeptide of the invention and,optionally, a pharmaceutically acceptable carrier, such as the diluentsphosphate buffered saline or a bicarbonate solution (e.g., 0.24 MNaHCO₃). The carriers used in the composition are selected on the basisof the mode and route of administration, and standard pharmaceuticalpractice. Suitable pharmaceutical carriers and diluents, as well aspharmaceutical necessities for their use, are described in Remington'sPharmaceutical Sciences. An adjuvant, e.g., a cholera toxin, Escherichiacoli heat-labile enterotoxin (LT), liposome, or immune-stimulatingcomplex (ISCOM), can also be included in the new vaccine composition, ifnecessary.

The amount of vaccine administered will depend, for example, on theparticular peptide antigen in the polypeptide, whether an adjuvant isco-administered with the antigen, the type of adjuvant co-administered,the mode and frequency of administration, and the desired effect (e.g.,protection or treatment), as can be determined by one skilled in theart. In general, the new vaccine antigens are administered in amountsranging between 1 μg and 100 mg polypeptide per adult human dose. Ifadjuvants are administered with the vaccines, amounts ranging between 1ng and 1 mg per adult human dose can generally be used. Administrationis repeated as necessary, as can be determined by one skilled in theart. For example, a priming dose can be followed by three booster dosesat weekly intervals. A booster shot can be given at 8 to 12 weeks afterthe first immunization, and a second booster can be given at 16 to 20weeks, using the same formulation. Sera or T-cells can be taken from theindividual for testing the immune response elicited by the vaccineagainst the neurotoxin. Methods of assaying antibodies or cytotoxic Tcells against a specific antigen are well known in the art. Additionalboosters can be given as needed. By varying the amount of polypeptide,the copy number of peptide antigen in the polypeptide, and frequency ofadministration, the immunization protocol can be optimized for elicitinga maximal immune response.

Before administering the above compositions in humans, toxicity andefficacy testing in animals are desirable. In an example of efficacytesting, mice (e.g., Swiss-Webster mice) can be vaccinated via an oralor parenteral route with a composition containing a polypeptide of theinvention. For vaccines against an infectious agent, after the initialvaccination or after optional booster vaccinations, the mice (andcorresponding control mice receiving mock vaccinations) are challengedwith a LD₉₅ dose of the infectious agent. End points other thanlethality can also be used. Efficacy is determined if mice receiving thevaccine die at a rate lower than the mock-vaccinated mice. Preferably,the difference in death rates should be statistically significant.Rabbits can be used in the above testing procedure instead of mice.

Alternatively, the new vaccine compositions can be administered asISCOMs. Protective immunity has been generated in a variety ofexperimental models of infection, including toxoplasmosis andEpstein-Barr virus-induced tumors, using ISCOMS as the delivery vehiclefor antigens (Mowat et al., Immunology Today 12:383-385, 1991). Doses ofantigen as low as 1 μg encapsulated in ISCOMs have been found to produceclass I mediated cytotoxic T cell responses (Takahashi et al., Nature344:873-875, 1990).

Without further elaboration, it is believed that one skilled in the artcan, based on the above disclosure and the description below, utilizethe present invention to its fullest extent. The following examples areto be construed as merely illustrative of how one skilled in the art canpractice the invention and are not limitative of the remainder of thedisclosure in any way. Any publications cited in this disclosure arehereby incorporated by reference.

EXAMPLE 1 A Multimeric Vaccine Against Gonadotropin Releasing Hormone

Gonadotropin releasing hormone (GnRH) is a decapeptide produced by thearcuate nuclei of the hypothalamus and regulates expression ofluteinizing hormone and follicle-stimulating hormone, which in turnregulates gonad development in humans. In addition, increased expressionof GnRH and its receptor has been correlated with a variety of tumors,including cancer of the breast, ovary, endometrium, and prostate. See,e.g., Imai et al., Cancer 74:2555-2561, 1994; Eidne et al., J. Clin.Endocrinol. Metab. 64:425-432, 1987; and Irmer et al., Cancer Res.55:817-822, 1995. Therefore, antibodies against GnRH may provide a meansto modulate reproductive hormone activity and/or cancer development andprogression.

To produce an antigen containing concatamers of GnRH fused to a receptorbinding domain, the following approach, as outlined in FIGS. 1-3, wasused. This procedure was designated template repeat PCR (TRPCR).

Two oligonucleotides were designed for TRPCR (FIG. 1). Oligo A encodedtarget antigen GnRH. Oligo B was complementary to oligo A. Oligo A isdissected into 5′ half A1 and 3′ half A2, both halves being of equallengths. Oligo B is dissected into 5′ half B1 and 3′ half B2, again bothhalves being of equal lengths. A1 was complementary to B1, while A2 wascomplementary to B2.

The thermal cycler was programmed for denaturation at 94° C. for 30seconds, annealing at 37° C. for 30 seconds, and extension at 72° C. for30 seconds. Using oligos A and B, PCR was performed for 30 cycles,followed by a final extension at 72° C. for 10 minutes. This PCR shouldproduced DNA species containing repeated sequences encoding GnRH, asillustrated in FIG. 2.

Two adapter primers (Adaptor A and Adaptor B) were designed to add anappropriate stop codon at the end of the repeated open reading frame(ORF) and two suitable restriction sites flanking the ORF (FIG. 3). Forthis step (which was designated adaptor PCR), the template for the PCRwas a 100-fold dilution of the TRPCR product produced as describedabove. The thermal cycler was programmed as described above, except thatthe denaturation was set for 1 minute instead of 30 seconds. Theresulting PCR product contained a SacII site at the 5′ end, an EcORIsite at the 3′ end, and a stop codon at the end of the ORF.

The products of TRPCR and adaptor-PCR were then examined on apolyacrylamide gel. The majority of the TRPCR products were 500 bp to700 bp in length. After adaptor PCR, products were distributed in aladder, the lowest band containing the dimer of the GnRH DNA repeat andthe slower migrating bands containing higher order multimers. The numberof repeats present ranged from 3 to at least 12. One clone containing 12repeats of GnRH coding sequence was chosen for further study.

The DNA fragment encoding 12 repeats of GnRH was subcloned into plasmidPPEDI, which was produced by subcloning the sequence encoding domain Iaof PE in pJH14 (Hwang et al., J. Biol. Chem. 264:2379-2384, 1989) intopET (Novagen). This plasmid expresses a polypeptide containing domain Iaof PE, which includes the toxin receptor binding domain of the toxin,and contains a His₆ tag at its N-terminus. The GnRH repeats wereinserted at the 3′ end of PE structure gene to produce pPEDIG12. Theprotein produced by this expression plasmid was designated PEIa-GnRH12.

pPEDIG12 was transformed into BL21(DE3)lysS. The transformants werecultured at 37° C. in LB medium containing ampicillin (50 μg/ml),chloramphenicol (25 μg/ml) and tetracycline (10 μg/ml). When the A₆₀₀ ofthe culture reached 0.2, IPTG was added to the culture to medium toachieve a final concentration of 0.1 mM. Cells were cultured for another90 minutes and then harvested. Since PEIa-GnRH12 is overexpressed in theform of inclusion bodies, the cells were extracted with 6 M urea.

The extracts were then purified using the Novagen pET His-Tag System.The fractions containing PEIa-GnRH12 were collected and dialysed against50 mM ammonium acetate (pH 4.0). PEIa-GnRH12 was stable for at least 6months when stored at 4° C. After nickel-agarose affinity columnchromatography, PEIa-GnRH12 was isolated to about 95% purity, asdetermined by SDS-PAGE.

To test this new immunogen, six week old New Zealand female rabbits wereimmunized with PEIa-GnRH12. A 0.5 ml bolus containing 100 μg ofPEIa-GnRH12 and 125 μg aluminum phosphate (pH 7.0) was injected into therabbit at the first time point (six weeks after birth). One week afterthe first immunization, an identical 0.5 ml bolus was injected into therabbits for a second immunization, followed by an identical injectiontwo weeks after the first immunization. After the three immunizations,sera were collected for ELISA and immunoblotting analysis. To testwhether antibodies against the multimeric antigen was directed tomonomeric epitopes or to epitopes spanning the junction betweenmonomers, several GST fusion proteins containing various multimers ofGnRH were produced as targets for ELISA.

The antibodies produced in the immunized rabbits failed to recognizewith GST alone, while the sera at 5,000-fold dilution recognized allGST-GnRH fusion proteins to the same extent, irrespectively of how manyGnRH repeats the GST fusion proteins contained. These results suggestedthat most of the antibodies produced in response to the immunogenrecognized monomeric GnRH epitopes rather than any hybrid epitopescreated by concatemerization.

GST-GnRH1 and GST-GnRH5 were specifically used as ELISA targets. Serafrom thrice immunized rabbits, diluted 10,000-fold, recognized GST-GnRH1and GST-GnRH5 to the same extent, thereby confirming that the immuneresponse against the immunogen was directed to GnRH monomer-specificepitopes.

To confirm that the antibodies elicited by the new immunogen wasphysiologically active, immunized rabbits were monitored for ovarydevelopment. As a control, rabbits were also immunized with PEIa-TopN8,a polypeptide containing domain Ia of PE fused to 8 repeats of a10-amino acid topoisomerase N-terminal peptide. The PEIa-TopN8-immunizedrabbits showed normal ovary development, while the ovaries ofPEIa-GnRH12-immunized rabbits were significantly decreased in size.Thus, the GnRH immunogen produced can be used as an immunogen to induceautoantibodies useful for treating GnRH-associated diseases.

To further confirm the utility of the new GnRH immunogen, mice and a pigwere also immunized with PEIa-GnRH12. The mice immunization regimen wasidentical to the rabbit immunization described above, except that eachmouse received a 100 μl bolus containing 10 μg PEIa-GnRH12 and 25 μgaluminum phosphate (pH 7.0) for each injection. In addition, a 24day-old pig was injection once with a 1 ml bolus containing 10 mgPEIa-GnRH12 and 250 μg aluminum phosphate (pH 7.0). GnRH-specificantibodies were readily elicited in the mice and pig, as well as inrabbits, indicating that the antigens can elicit an immune response in avariety of animals. Further, a single immunization was sufficient toelicit an immune response.

EXAMPLE 2 A Multimeric Vaccine Against Vaccinia Virus

Instead of a GnRH peptide sequence, a DNA sequence containing a repeatof the vaccinia virus coat protein peptide LIGICVAVTVAI (SEQ ID NO:2)was constructed using PCR as described in Example 1 above. A PE fusionprotein containing 16 repeats of the virus coat protein peptide wasproduced and purified according to the procedures in Example 1.

Six-week old rabbits were injected with a 1 ml bolus containing 100 μgviral peptide repeat antigen and 100 μg complete Freund's adjuvant. Fourweeks later, the rabbits received a second 1 ml injection containing 100μg antigen and 100 μg incomplete Freund's adjuvant. At 8 weeks after thefirst immunization, the rabbits were injected with a 1 ml boluscontaining 100 μg antigen (no adjuvant). Rabbits thrice immunized withthe virus coat protein immunogen produced vaccinia virus-specificantibodies.

Thus, the general procedure of linking a peptide repeat to a PE receptorbinding domain was shown to be successful for a second peptide.

Other Embodiments

It is to be understood that while the invention has been described inconjunction with the detailed description thereof, the foregoingdescription is intended to illustrate and not limit the scope of theinvention, which is defined by the scope of the appended claims. Otheraspects, advantages, and modifications are within the scope of thisinvention.

1. An isolated nucleic acid encoding a polypeptide, wherein thepolypeptide comprises (1) a Pseudomonas exotoxin A fragment consistingof the receptor binding domain of the Pseudomonas exotoxin A and (2) atleast two copies of an antigenic peptide sequence wherein said copiesoccur as a single block of peptide repeats without intervening aminoacids in between any two copies.
 2. An isolated nucleic acid encoding apolypeptide, wherein the polypeptide comprises (1) a Pseudomonasexotoxin A fragment consisting of the receptor binding domain of aPseudomonas exotoxin A and (2) at least two copies of an antigenicpeptide sequence comprising SEQ ID NO:1, and has no toxicity.
 3. Anisolated nucleic acid encoding a polypeptide, wherein the polypeptidecomprises (1) a Pseudomonas exotoxin A fragment consisting of thereceptor binding domain of a Pseudomonas exotoxin A and (2) 10 to 20copies of an antigenic peptide sequence, and has no toxicity.
 4. Anisolated nucleic acid encoding a polypeptide, wherein the polypeptidecomprises (1) a Pseudomonas exotoxin A fragment consisting of thereceptor binding domain of a Pseudomonas exotoxin A and (2) at least twocopies of an antigenic peptide sequence in a consecutive series, and hasno toxicity.